The only naturally occurring nonsense suppressor tRNAs described in higher eukaryotes are two opal suppressor serine tRNAs that occur in vertebrate tissues. These tRNAs have several unique features: (1) they are 90 nucleotides in length and thus are the longest tRNAs sequenced to date; (2) they are phosphorylated on their serine moiety to form phosphoseryl- tRNA; (3) they have few modified bases compared to other tRNAs; (4) they are encoded by a single gene even though several pyrimidine transitions occur post-transcriptionally and one of the transitions occurs in the anticodon; and (5) the primary transcript arises, unlike any other known tRNA, without processing on the 5' side of the gene product. Among animals, the gene occurs in members of the Phyla Chlordata (tunicate, amphioxus, lamprey, hag fish, horned shark, winter flounder, Xenopus, chicken and bovine), Arthropoda, Mollusca, Aschelminthes and Porifera. The gene was also detected in the genomes of representatives from the Monera, Plant and Protist Kingdoms. The genes encoding the opal suppressor tRNAs which have been isolated and sequenced from human, rabbit, chicken and Xenopus genomes are transcribed in vivo in Xenopus oocytes and are transcribed in vitro in HeLa cell extracts. Fingerprints of the processed transcript from the Xenopus gene show that the gene is faithfully transcribed and that initiation of transcription occurs at the first nucleotide within the gene. The 3' trailer sequence is removed by purified 3' processing enzyme. The triphosphate on the 5' nucleotide is preserved in transport of the gene product from the nucleus to the cytoplasm and remains intact in the cytoplasm, suggesting that it may have a function on the mature tRNA. The gene was mapped to human chromosome 19 and the corresponding pseudogene to human chromosome 21.